Improved reference genome of Aedes aegypti informs arbovirus vector control.

Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.

Molecular drivers of insecticide resistance in the Sahelo-Sudanian populations of a major malaria vector Anopheles coluzzii.

BACKGROUND: Information on common markers of metabolic resistance in malaria vectors from countries sharing similar eco-climatic characteristics can facilitate coordination of malaria control. Here, we characterized populations of the major malaria vector Anopheles coluzzii from Sahel region, spanning four sub-Saharan African countries: Nigeria, Niger, Chad and Cameroon. RESULTS: Genome-wide transcriptional analysis identified major genes previously implicated in pyrethroid and/or cross-resistance to other insecticides, overexpressed across the Sahel, including CYP450s, glutathione S-transferases, carboxylesterases and cuticular proteins. Several, well-known markers of insecticide resistance were found in high frequencies-including in the voltage-gated sodium channel (V402L, I940T, L995F, I1527T and N1570Y), the acetylcholinesterase-1 gene (G280S) and the CYP4J5-L43F (which is fixed). High frequencies of the epidemiologically important chromosomal inversion polymorphisms, 2La, 2Rb and 2Rc, were observed (~80% for 2Rb and 2Rc). The 2La alternative arrangement is fixed across the Sahel. Low frequencies of these inversions (<10%) were observed in the fully insecticide susceptible laboratory colony of An. coluzzii (Ngoussou). Several of the most commonly overexpressed metabolic resistance genes sit in these three inversions. Two commonly overexpressed genes, GSTe2 and CYP6Z2, were functionally validated. Transgenic Drosophila melanogaster flies expressing GSTe2 exhibited extremely high DDT and permethrin resistance (mortalities <10% in 24h). Serial deletion of the 5' intergenic region, to identify putative nucleotide(s) associated with GSTe2 overexpression, revealed that simultaneous insertion of adenine nucleotide and a transition (T->C), between Forkhead box L1 and c-EST putative binding sites, were responsible for the high overexpression of GSTe2 in the resistant mosquitoes. Transgenic flies expressing CYP6Z2 exhibited marginal resistance towards 3-phenoxybenzylalcohol (a primary product of pyrethroid hydrolysis by carboxylesterases) and a type II pyrethroid, α-cypermethrin. However, significantly higher mortalities were observed in CYP6Z2 transgenic flies compared with controls, on exposure to the neonicotinoid, clothianidin. This suggests a possible bioactivation of clothianidin into a toxic intermediate, which may make it an ideal insecticide against populations of An. coluzzii overexpressing this P450. CONCLUSIONS: These findings will facilitate regional collaborations within the Sahel region and refine implementation strategies through re-focusing interventions, improving evidence-based, cross-border policies towards local and regional malaria pre-elimination.

An Africa-wide genomic evolution of insecticide resistance in the malaria vector Anopheles funestus involves selective sweeps, copy number variations, gene conversion and transposons.

Insecticide resistance in malaria vectors threatens to reverse recent gains in malaria control. Deciphering patterns of gene flow and resistance evolution in malaria vectors is crucial to improving control strategies and preventing malaria resurgence. A genome-wide survey of Anopheles funestus genetic diversity Africa-wide revealed evidences of a major division between southern Africa and elsewhere, associated with different population histories. Three genomic regions exhibited strong signatures of selective sweeps, each spanning major resistance loci (CYP6P9a/b, GSTe2 and CYP9K1). However, a sharp regional contrast was observed between populations correlating with gene flow barriers. Signatures of complex molecular evolution of resistance were detected with evidence of copy number variation, transposon insertion and a gene conversion between CYP6P9a/b paralog genes. Temporal analyses of samples before and after bed net scale up suggest that these genomic changes are driven by this control intervention. Multiple independent selective sweeps at the same locus in different parts of Africa suggests that local evolution of resistance in malaria vectors may be a greater threat than trans-regional spread of resistance haplotypes.

Multi-omics analysis identifies a CYP9K1 haplotype conferring pyrethroid resistance in the malaria vector Anopheles funestus in East Africa.

Metabolic resistance to pyrethroids is a menace to the continued effectiveness of malaria vector controls. Its molecular basis is complex and varies geographically across Africa. Here, we used a multi-omics approach, followed-up with functional validation to show that a directionally selected haplotype of a cytochrome P450, CYP9K1 is a major driver of resistance in Anopheles funestus. A PoolSeq GWAS using mosquitoes alive and dead after permethrin exposure, from Malawi and Cameroon, detected candidate genomic regions, but lacked consistency across replicates. Targeted sequencing of candidate resistance genes detected several SNPs associated with known pyrethroid resistance QTLs. The most significant SNPs were in the cytochrome P450 CYP304B1 (Cameroon), CYP315A1 (Uganda) and the ABC transporter gene ABCG4 (Malawi). However, when comparing field resistant mosquitoes to laboratory susceptible, the pyrethroid resistance locus rp1 and SNPs around the ABC transporter ABCG4 were consistently significant, except for Uganda where SNPs in the P450 CYP9K1 was markedly significant. In vitro heterologous metabolism assays with recombinant CYP9K1 revealed that it metabolises type II pyrethroid (deltamethrin; 64% depletion) but not type I (permethrin; 0%), while moderately metabolising DDT (17%). CYP9K1 exhibited reduced genetic diversity in Uganda underlying an extensive selective sweep. Furthermore, a glycine to alanine (G454A) amino acid change in CYP9K1 was fixed in Ugandan mosquitoes but not in other An. funestus populations. This study sheds further light on the evolution of metabolic resistance in a major malaria vector by implicating more genes and variants that can be used to design field-applicable markers to better track resistance Africa-wide.

Characterization of the glutathione S-transferase genes in the sand flies Phlebotomus papatasi and Lutzomyia longipalpis shows expansion of the novel glutathione S-transferase xi (X) class.

Leishmaniasis control often relies upon insecticidal control of phlebotomine sandfly vector populations. Such methods are vulnerable to the evolution of insecticide resistance via a range of molecular mechanisms. There is evidence that two major resistance mechanisms, target site insensitivity and metabolic resistance, have evolved in some sandfly populations and further genetic characterization of resistance would be useful to understand and combat it. To facilitate the study of the mechanisms of metabolic resistance, here we improved the annotation and characterized a major detoxification gene family, the glutathione-s-transferases (GST), in the genomes of two sand fly species: Phlebotomus papatasi and Lutzomyia longipalpis. The compositions of the GST gene family differ markedly from those of Aedes and Anopheles mosquitoes. Most strikingly, the xi (X) class of GSTs appears to have expanded in both sand fly genomes. Our results provide a basis for further studies of metabolic resistance mechanisms in these important disease vector species.

A 6.5-kb intergenic structural variation enhances P450-mediated resistance to pyrethroids in malaria vectors lowering bed net efficacy.

Elucidating the complex evolutionary armory that mosquitoes deploy against insecticides is crucial to maintain the effectiveness of insecticide-based interventions. Here, we deciphered the role of a 6.5-kb structural variation (SV) in driving cytochrome P450-mediated pyrethroid resistance in the malaria vector, Anopheles funestus. Whole-genome pooled sequencing detected an intergenic 6.5-kb SV between duplicated CYP6P9a/b P450s in pyrethroid-resistant mosquitoes through a translocation event. Promoter analysis revealed a 17.5-fold higher activity (p < .0001) for the SV- carrying fragment than the SV- free one. Quantitative real-time PCR expression profiling of CYP6P9a/b for each SV genotype supported its role as an enhancer because SV+/SV+ homozygote mosquitoes had a significantly greater expression for both genes than heterozygotes SV+/SV- (1.7- to 2-fold) and homozygotes SV-/SV- (4-to 5-fold). Designing a PCR assay revealed a strong association between this SV and pyrethroid resistance (SV+/SV+ vs. SV-/SV-; odds ratio [OR] = 2,079.4, p < .001). The 6.5-kb SV is present at high frequency in southern Africa (80%-100%) but absent in East/Central/West Africa. Experimental hut trials revealed that homozygote SV mosquitoes had a significantly greater chance to survive exposure to pyrethroid-treated nets (OR 27.7; p < .0001) and to blood feed than susceptible mosquitoes. Furthermore, mosquitoes homozygote-resistant at the three loci (SV+/CYP6P9a_R/CYP6P9b_R) exhibited a higher resistance level, leading to a far superior ability to survive exposure to nets than those homozygotes susceptible at the three loci, revealing a strong additive effect. This study highlights the important role of structural variations in the development of insecticide resistance in malaria vectors and their detrimental impact on the effectiveness of pyrethroid-based nets.

Genomic Footprints of Selective Sweeps from Metabolic Resistance to Pyrethroids in African Malaria Vectors Are Driven by Scale up of Insecticide-Based Vector Control.

Insecticide resistance in mosquito populations threatens recent successes in malaria prevention. Elucidating patterns of genetic structure in malaria vectors to predict the speed and direction of the spread of resistance is essential to get ahead of the 'resistance curve' and to avert a public health catastrophe. Here, applying a combination of microsatellite analysis, whole genome sequencing and targeted sequencing of a resistance locus, we elucidated the continent-wide population structure of a major African malaria vector, Anopheles funestus. We identified a major selective sweep in a genomic region controlling cytochrome P450-based metabolic resistance conferring high resistance to pyrethroids. This selective sweep occurred since 2002, likely as a direct consequence of scaled up vector control as revealed by whole genome and fine-scale sequencing of pre- and post-intervention populations. Fine-scaled analysis of the pyrethroid resistance locus revealed that a resistance-associated allele of the cytochrome P450 monooxygenase CYP6P9a has swept through southern Africa to near fixation, in contrast to high polymorphism levels before interventions, conferring high levels of pyrethroid resistance linked to control failure. Population structure analysis revealed a barrier to gene flow between southern Africa and other areas, which may prevent or slow the spread of the southern mechanism of pyrethroid resistance to other regions. By identifying a genetic signature of pyrethroid-based interventions, we have demonstrated the intense selective pressure that control interventions exert on mosquito populations. If this level of selection and spread of resistance continues unabated, our ability to control malaria with current interventions will be compromised.

Experimental swap of Anopheles gambiae's assortative mating preferences demonstrates key role of X-chromosome divergence island in incipient sympatric speciation.

Although many theoretical models of sympatric speciation propose that genes responsible for assortative mating amongst incipient species should be associated with genomic regions protected from recombination, there are few data to support this theory. The malaria mosquito, Anopheles gambiae, is known for its sympatric cryptic species maintained by pre-mating reproductive isolation and its putative genomic islands of speciation, and is therefore an ideal model system for studying the genomic signature associated with incipient sympatric speciation. Here we selectively introgressed the island of divergence located in the pericentric region of the X chromosome of An. gambiae s.s. into its sister taxon An. coluzzii through 5 generations of backcrossing followed by two generations of crosses within the introgressed strains that resulted in An. coluzzii-like recombinant strains fixed for the M and S marker in the X chromosome island. The mating preference of recombinant strains was then tested by giving virgin recombinant individuals a choice of mates with X-islands matching and non-matching their own island type. We show through genetic analyses of transferred sperm that recombinant females consistently mated with matching island-type males thereby associating assortative mating genes with the X-island of divergence. Furthermore, full-genome sequencing confirmed that protein-coding differences between recombinant strains were limited to the experimentally swapped pericentromeric region. Finally, targeted-genome comparisons showed that a number of these unique differences were conserved in sympatric field populations, thereby revealing candidate speciation genes. The functional demonstration of a close association between speciation genes and the X-island of differentiation lends unprecedented support to island-of-speciation models of sympatric speciation facilitated by pericentric recombination suppression.

Molecular tools for studying the major malaria vector Anopheles funestus: improving the utility of the genome using a comparative poly(A) and Ribo-Zero RNAseq analysis.

BACKGROUND: Next-generation sequencing (NGS) offers great opportunities for studying the biology of insect vectors of disease. Prerequisites for successful analyses include high quality annotated genome assemblies and that techniques designed for use with model organisms be tested and optimised for use with these insects. We aimed to test and improve genomic tools for studying the major malaria vector Anopheles funestus. RESULTS: To guide future RNAseq transcriptomic studies of An. funestus, we compared two methods for enrichment of non-ribosomal RNA for analysis: enrichment of polyadenylated RNA and ribosomal RNA depletion using a kit designed to deplete human/rat/mouse rRNA. We found large differences between the two methods in the resulting transcriptomes, some of which is due to differential representation of polyadenylated and non-polyadenylated transcripts. We used the RNAseq data for validation and targeted manual editing of the draft An. funestus genome annotation, validating 62 % of annotated introns, manually improving the annotation of seven gene families involved in the detoxification of xenobiotics and integrated two published transcriptomic datasets with the recently published genome assembly. CONCLUSIONS: The mRNA enrichment method makes a substantial, replicable difference to the transcriptome composition, at least partly due to the representation of non-polyadenylated transcripts in the final transcriptome. Therefore, great care should be taken in comparing gene expression data among studies. Ribosomal RNA depletion of total RNA using a kit designed to deplete human/rat/mouse rRNA works in mosquitoes and, we argue, results in a truer representation of the transcriptome than poly(A) selection. The An. funestus genome annotation can be considerably improved with the help of these new RNAseq data and further guided manual gene editing efforts will be of great benefit to the Anopheles research community for studies of this insect's genome and transcriptome.

Rise of multiple insecticide resistance in Anopheles funestus in Malawi: a major concern for malaria vector control.

BACKGROUND: Deciphering the dynamics and evolution of insecticide resistance in malaria vectors is crucial for successful vector control. This study reports an increase of resistance intensity and a rise of multiple insecticide resistance in Anopheles funestus in Malawi leading to reduced bed net efficacy. METHODS: Anopheles funestus group mosquitoes were collected in southern Malawi and the species composition, Plasmodium infection rate, susceptibility to insecticides and molecular bases of the resistance were analysed. RESULTS: Mosquito collection revealed a predominance of An. funestus group mosquitoes with a high hybrid rate (12.2 %) suggesting extensive species hybridization. An. funestus sensu stricto was the main Plasmodium vector (4.8 % infection). Consistently high levels of resistance to pyrethroid and carbamate insecticides were recorded and had increased between 2009 and 2014. Furthermore, the 2014 collection exhibited multiple insecticide resistance, notably to DDT, contrary to 2009. Increased pyrethroid resistance correlates with reduced efficacy of bed nets (<5 % mortality by Olyset(®) net), which can compromise control efforts. This change in resistance dynamics is mirrored by prevalent resistance mechanisms, firstly with increased over-expression of key pyrethroid resistance genes (CYP6Pa/b and CYP6M7) in 2014 and secondly, detection of the A296S-RDL dieldrin resistance mutation for the first time. However, the L119F-GSTe2 and kdr mutations were absent. CONCLUSIONS: Such increased resistance levels and rise of multiple resistance highlight the need to rapidly implement resistance management strategies to preserve the effectiveness of existing insecticide-based control interventions.

Sexual reproduction and genetic exchange in parasitic protists.

A key part of the life cycle of an organism is reproduction. For a number of important protist parasites that cause human and animal disease, their sexuality has been a topic of debate for many years. Traditionally, protists were considered to be primitive relatives of the 'higher' eukaryotes, which may have diverged prior to the evolution of sex and to reproduce by binary fission. More recent views of eukaryotic evolution suggest that sex, and meiosis, evolved early, possibly in the common ancestor of all eukaryotes. However, detecting sex in these parasites is not straightforward. Recent advances, particularly in genome sequencing technology, have allowed new insights into parasite reproduction. Here, we review the evidence on reproduction in parasitic protists. We discuss protist reproduction in the light of parasitic life cycles and routes of transmission among hosts.

Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii.

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S-transferases. RNA-seq-based genome-wide transcriptional analyses supported by quantitative PCR identified glutathione S-transferase, GSTe2 (RNA-seqFC = 2.93 and qRT-PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA-seqFC = 2.39 and qRT-PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid-resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace-1 mutation was detected in bendiocarb-resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon.

Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters.

BACKGROUND: The filamentous fungus Aspergillus nidulans has been a tractable model organism for cell biology and genetics for over 60 years. It is among a large number of Aspergilli whose genomes have been sequenced since 2005, including medically and industrially important species. In order to advance our knowledge of its biology and increase its utility as a genetic model by improving gene annotation we sequenced the transcriptome of A. nidulans with a focus on 5' end analysis. RESULTS: Strand-specific whole transcriptome sequencing showed that 80-95% of annotated genes appear to be expressed across the conditions tested. We estimate that the total gene number should be increased by approximately 1000, to 11,800. With respect to splicing 8.3% of genes had multiple alternative transcripts, but alternative splicing by exon-skipping was very rare. 75% of annotated genes showed some level of antisense transcription and for one gene, meaB, we demonstrated the antisense transcript has a regulatory role. Specific sequencing of the 5' ends of transcripts was used for genome wide mapping of transcription start sites, allowing us to interrogate over 7000 promoters and 5' untranslated regions. CONCLUSIONS: Our data has revealed the complexity of the A. nidulans transcriptome and contributed to improved genome annotation. The data can be viewed on the AspGD genome browser.

The Colorectal Cancer Gut Environment Regulates Activity of the Microbiome and Promotes the Multidrug Resistant Phenotype of ESKAPE and Other Pathogens.

Taxonomic composition of the gut microbiota in colorectal cancer (CRC) patients is altered, a newly recognized driving force behind the disease, the activity of which has been overlooked. We conducted a pilot study on active microbial taxonomic composition in the CRC gut via metatranscriptome and 16S rRNA gene (rDNA) sequencing. We revealed sub-populations in CRC (n = 10) and control (n = 10) cohorts of over-active and dormant species, as changes in activity were often independent from abundance. Strikingly, the diseased gut significantly influenced transcription of butyrate producing bacteria, clinically relevant ESKAPE, oral, and Enterobacteriaceae pathogens. A focused analysis of antibiotic (AB) resistance genes showed that both CRC and control microbiota displayed a multidrug resistant phenotype, including ESKAPE species. However, a significant majority of AB resistance determinants of several AB families were upregulated in the CRC gut. We found that environmental gut factors regulated AB resistance gene expression in vitro of aerobic CRC microbiota, specifically acid, osmotic, and oxidative pressures in a predominantly health-dependent manner. This was consistent with metatranscriptome analysis of these cohorts, while osmotic and oxidative pressures induced differentially regulated responses. This work provides novel insights into the organization of active microbes in CRC, and reveals significant regulation of functionally related group activity, and unexpected microbiome-wide upregulation of AB resistance genes in response to environmental changes of the cancerous gut. IMPORTANCE The human gut microbiota in colorectal cancer patients have a distinct population compared to heathy counterparts. However, the activity (gene expression) of this community has not been investigated. Following quantification of both expressed genes and gene abundance, we established that a sub-population of microbes lies dormant in the cancerous gut, while other groups, namely, clinically relevant oral and multi-drug resistant pathogens, significantly increased in activity. Targeted analysis of community-wide antibiotic resistance determinants found that their expression occurs independently of antibiotic treatment, regardless of host health. However, its expression in aerobes, in vitro, can be regulated by specific environmental stresses of the gut, including organic and inorganic acid pressure in a health-dependent manner. This work advances the field of microbiology in the context of disease, showing, for the first time, that colorectal cancer regulates activity of gut microorganisms and that specific gut environmental pressures can modulate their antibiotic resistance determinants expression.

The Colorectal Cancer Microbiota Alter Their Transcriptome To Adapt to the Acidity, Reactive Oxygen Species, and Metabolite Availability of Gut Microenvironments.

The gut microbiome is implicated in the pathology of colorectal cancer (CRC). However, the mechanisms by which the microbiota actively contribute to disease onset and progression remain elusive. In this pilot study, we sequenced fecal metatranscriptomes of 10 non-CRC and 10 CRC patient gut microbiomes and conducted differential gene expression analyses to assess any changed functionality in disease. We report that oxidative stress responses were the dominant activity across cohorts, an overlooked protective housekeeping role of the human gut microbiome. However, expression of hydrogen peroxide and nitric oxide-scavenging genes was diminished and augmented, respectively, positing that these regulated microbial responses have implications for CRC pathology. CRC microbes enhanced expression of genes for host colonization, biofilm formation, genetic exchange, virulence determinants, antibiotic, and acid resistances. Moreover, microbes promoted transcription of genes involved in metabolism of several beneficial metabolites, suggesting their contribution to patient metabolite deficiencies previously solely attributed to tumor cells. We showed in vitro that expression of genes involved in amino acid-dependent acid resistance mechanisms of meta-gut Escherichia coli responded differently to acid, salt, and oxidative pressures under aerobic conditions. These responses were mostly dictated by the host health status of origin of the microbiota, suggesting their exposure to fundamentally different gut conditions. These findings for the first time highlight mechanisms by which the gut microbiota can either protect against or drive colorectal cancer and provide insights into the cancerous gut environment that drives functional characteristics of the microbiome. IMPORTANCE The human gut microbiota has the genetic potential to drive colorectal cancer onset and progression; however, the expression of this genetic potential during the disease has not been investigated. We found that microbial expression of genes that detoxify DNA-damaging reactive oxygen species, which drive colorectal cancer, is compromised in cancer. We observed a greater activation of expression of genes involved in virulence, host colonization, exchange of genetic material, metabolite utilization, defense against antibiotics, and environmental pressures. Culturing gut Escherichia coli of cancerous and noncancerous metamicrobiota revealed different regulatory responses of amino acid-dependent acid resistance mechanisms in a health-dependent manner under environmental acid, oxidative, and osmotic pressures. Here, for the first time, we demonstrate that the activity of microbial genomes is regulated by the health status of the gut in vivo and in vitro and provides new insights for shifts in microbial gene expression in colorectal cancer.

A parasitic nematode induces dysbiosis in susceptible but not resistant gastropod hosts.

Animals' gut microbiomes affect a wide array of biological processes including immunity and protection from pathogens. However, how the microbiome changes due to infection by parasites is still largely unknown, as is how the microbiome changes in hosts that differ in their susceptibility to parasites. To investigate this, we exposed two slug species of differing susceptibility to the parasitic nematode Phasmarhabditis hermaphrodita (Deroceras reticulatum is highly susceptible and Ambigolimax valentianus resistant to the nematode) and profiled the gut microbiota after 7 and 14 days. Before infection, both slug species' microbiota was dominated by similar bacterial genera: Pseudomonas (by far the most abundant), Sphingobacterium, Pedobacter, Chryseobacterium, and Flavobacterium. In the resistant host A. valentianus, there was no significant change in the bacterial genera after infection, but in D. reticulatum, the bacterial profile changed, with a decrease in the abundance of Pseudomonadaceae and an increase in the abundance of Flavobacteriaceae and Sphingobacteriaceae after 7 days postinfection. This suggests nematode infection causes dysbiosis in hosts that are susceptible to infection, but the microbiome of resistant species remains unaltered. In summary, the regulation of the immune system is tightly linked with host survival, and nematode infection can alter the microbiome structure.

Case Report: Simple Nodular Cutaneous Leishmaniasis Caused by Autochthonous Leishmania (Mundinia) orientalis in an 18-Month-Old Girl: The First Pediatric Case in Thailand and Literature Review.

We report an autochthonous case of simple, localized cutaneous leishmaniasis in a healthy 18-month-old girl from southern Thailand. The patient presented with a solitary chronic cutaneous nodular lesion on her left cheek for approximately 1 year. Histopathological dissection of the cheek skin biopsy demonstrated remarkably nodular and interstitial infiltrates of lymphocytes and histiocytes full of intracellular oval-shaped amastigotes, consistent with cutaneous leishmaniasis. The Leishmania promastigotes were also cultured successfully from the lesion biopsy and were designated with the WHO code MHOM/TH/2021/CULE5. Using internal transcribed spacer 1-specific polymerase chain reaction, the parasite DNA was demonstrated in both saliva and lesion biopsy. Based on the BLASTn and phylogenetic analysis, the parasite was identified as Leishmania orientalis, clustered in the Mundinia subgenus. The patient responded well to a 6-week course of oral itraconazole, without recurrence. To our knowledge, this is the fourth case of autochthonous leishmaniasis resulting from L. orientalis and the youngest patient of leishmaniasis ever reported in Thailand. More importantly, we also demonstrate the clinical course of the lesion according to the timeline before and after treatment, which can help physicians better understand and provide an accurate diagnosis with appropriate treatment of this emerging parasitic disease.

A Multilocus Sequence Typing System (MLST) reveals a high level of diversity and a genetic component to Entamoeba histolytica virulence.

BACKGROUND: The outcome of an Entamoeba histolytica infection is variable and can result in either asymptomatic carriage, immediate or latent disease (diarrhea/dysentery/amebic liver abscess). An E. histolytica multilocus genotyping system based on tRNA gene-linked arrays has shown that genetic differences exist among parasites isolated from patients with different symptoms however, the tRNA gene-linked arrays cannot be located in the current assembly of the E. histolytica Reference genome (strain HM-1:IMSS) and are highly variable. RESULTS: To probe the population structure of E. histolytica and identify genetic markers associated with clinical outcome we identified in E. histolytica positive samples selected single nucleotide polymorphisms (SNPs) by multiplexed massive parallel sequencing. Profile SNPs were selected which, compared to the reference strain HM-1:IMSS sequence, changed an encoded amino acid at the SNP position, and were present in independent E. histolytica isolates from different geographical origins. The samples used in this study contained DNA isolated from either xenic strains of E. histolytica trophozoites established in culture or E. histolytica positive clinical specimens (stool and amebic liver abscess aspirates). A record of the SNPs present at 16 loci out of the original 21 candidate targets was obtained for 63 of the initial 84 samples (63% of asymptomatically colonized stool samples, 80% of diarrheal stool, 73% of xenic cultures and 84% of amebic liver aspirates). The sequences in all the 63 samples both passed sequence quality control metrics and also had the required greater than 8X sequence coverage for all 16 SNPs in order to confidently identify variants. CONCLUSIONS: Our work is in agreement with previous findings of extensive diversity among E. histolytica isolates from the same geographic origin. In phylogenetic trees, only four of the 63 samples were able to group in two sets of two with greater than 50% confidence. Two SNPs in the cylicin-2 gene (EHI_080100/XM_001914351) were associated with disease (asymptomatic/diarrhea p = 0.0162 or dysentery/amebic liver abscess p = 0.0003). This study demonstrated that there are genetic differences between virulent and avirulent E. histolytica strains and that this approach has the potential to define genetic changes that influence infection outcomes.

Microbiome Analysis of Malacopathogenic Nematodes Suggests No Evidence of a Single Bacterial Symbiont Responsible for Gastropod Mortality.

Nematodes and bacteria are prevalent in soil ecosystems, and some have evolved symbiotic relationships. In some cases, symbionts carry out highly specialized functions: a prime example being entomopathogenic nematodes (EPNs), which vector bacteria (Xenorhabdus or Photorhabdus) into insect hosts, killing them to provide a food source for the nematodes. It is thought that the commercially available malacopathogenic (kills slugs and snails) biocontrol nematode Phasmarhabditis hermaphrodita vectors a bacterium (Moraxella osloensis) into slugs to kill them. To investigate this further we used a metagenomic approach to profile the bacteria present in the commercial strain of P. hermaphrodita, a wild strain of P. hermaphrodita and two other Phasmarhabditis species (P. californica and P. neopapillosa), after they had killed their slug host (Deroceras invadens). We show that these nematodes do not exclusively associate with one bacterium but a range of species, with members of the phyla Pseudomonadota, Bacillota, Actinobacteriota and Bacteroidota the most prevalent. The commercial strain of P. hermaphrodita had the least diverse bacterial community. Furthermore, we found that the bacterium P. hermaphrodita has been cultured on for 25 years is not the expected species M. osloensis but is Psychrobacter spp. and the only strain of the Phasmarhabditis species to associate with Psychrobacter spp. was the commercial strain of P. hermaphrodita. In summary, we found no evidence to show that P. hermaphrodita rely exclusively on one bacterium to cause host mortality but found variable and diverse bacterial communities associated with these nematodes in their slug hosts.

Gene Conversion Explains Elevated Diversity in the Immunity Modulating APL1 Gene of the Malaria Vector Anopheles funestus.

Leucine-rich repeat proteins and antimicrobial peptides are the key components of the innate immune response to Plasmodium and other microbial pathogens in Anopheles mosquitoes. The APL1 gene of the malaria vector Anopheles funestus has exceptional levels of non-synonymous polymorphism across the range of An. funestus, with an average πn of 0.027 versus a genome-wide average of 0.002, and πn is consistently high in populations across Africa. Elevated APL1 diversity was consistent between the independent pooled-template and target-enrichment datasets, however no link between APL1 diversity and insecticide resistance was observed. Although lacking the diversity of APL1, two further mosquito innate-immunity genes of the gambicin anti-microbial peptide family had πn/πs ratios greater than one, possibly driven by either positive or balancing selection. The cecropin antimicrobial peptides were expressed much more highly than other anti-microbial peptide genes, a result discordant with current models of anti-microbial peptide activity. The observed APL1 diversity likely results from gene conversion between paralogues, as evidenced by shared polymorphisms, overlapping read mappings, and recombination events among paralogues. In conclusion, we hypothesize that higher gene expression of APL1 than its paralogues is correlated with a more open chromatin formation, which enhances gene conversion and elevated diversity at this locus.